Abstract Scrub typhus is a life-threatening zoonosis caused by Orientia tsutsugamushi organisms that are transmitted by the larvae of trombiculid mites. About one third of the world?s population is at risk of infection. However, the mechanism of bacterial dissemination from the skin and the reason that innate immune cells fail to control local infection are unknown; it is technically challenging to track the bacteria during infection in vivo. This project will use our newly established intradermal (i.d.) infection mouse model and fluorescence-labeled bacteria to define the mechanisms of bacterial dissemination. Our preliminary mouse data revealed containment and survival of CFSE-labeled Orientia in neutrophils for at least 12 hours in vitro. We also found neutrophils as the major infiltrating cell population responding to inactivated Orientia in the mouse skin. Given that neutrophil depletion via antibodies promoted mouse survival against acute infection in a lethal infection (i.v. infection model), we want to test the hypothesis that Orientia bacteria can hijack and utilize neutrophils as Trojan horses against host bactericidal activities, allowing bacterial dissemination from the initial skin infection sites to visceral organs. Specific Aim 1 will examine the dynamics of myeloid cell recruitment in the skin and the bacterial load in unique cell populations using multi-color flow cytometry. We will also evaluate effects of Orientia infection on neutrophil bactericidal activity, including reactive oxygen species generation, neutrophil extracellular trap formation, and granules/cytokine production both in vitro and in vivo. Specific Aim 2 will define neutrophil- mediated Orientia dissemination and immunopathogenesis. We will track CFSE-labeled Orientia among various organs (e.g. draining lymph nodes, bone marrow, lung, and spleen) after i.d. inoculation. Cellular markers of reverse migration will also be examined. To further validate the pathogenic role of neutrophils during disease progression, we will modulate neutrophils migration using selective and potent antagonists. This work will provide new insights into the innate immune determinants of Orientia infection and will lay the groundwork for our long-term goal of developing promising therapeutic strategies for this and other vector- borne diseases that share a common route of pathogen dissemination.